An Aptamer-Based Near-Infrared Fluorescence Nanoprobe for

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An Aptamer-Based Near-Infrared Fluorescence Nanoprobe for Detecting and Imaging of PLN Micropeptide in Cardiomyocytes Renhui Zhan, Xiaofeng Li, Wenfei Guo, Xiaojun Liu, zhi-xian Liu, Kehua Xu, and Bo Tang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.9b00026 • Publication Date (Web): 19 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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An Aptamer-Based Near-Infrared Fluorescence Nanoprobe for Detecting and Imaging of PLN Micropeptide in Cardiomyocytes Renhui Zhan†,‡ , Xiaofeng Li†, Wenfei Guo†, Xiaojun Liu†, Zhixian Liu†, Kehua Xu*,†, and Bo Tang*,† College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Shandong Normal University, Jinan 250014, P. R. China. ‡Medicine & Pharmacy Research Center, Binzhou Medical University, Shandong, Yantai 264003, P. R. China. Keywords: Heart failure, Micropeptide, Phospholamban, Aptamer, Nanoprobe, Fluorescence Imaging, biological detection †

ABSTRACT: A growing body of evidence indicates that micropeptides encoded by long noncoding RNAs (lncRNAs) act independently or as regulators of larger proteins in fundamental biological processes, especially in the maintenance of cellular homeostasis. However, due to their small size and low intracellular expression, visual monitoring of micropeptides in living cells is still a challenge. In this work, we have designed and synthesized an aptamer-based near-infrared fluorescence nanoprobe for fluorescence imaging of phospholamban (PLN), which is an intracellular micropeptide that affects calcium homeostasis, and is closely associated with human heart failure in clinic. The nanoprobe could respond specifically to PLN with excellent selectivity, high sensitivity, good nuclease stability and biocompatibility, and it was successfully applied for imaging of changes in PLN levels in cardiomyocytes and in frozen section of heart tissues. And further combined with clinical myocardial biopsy, we believe that the developed nanoprobe should be of great significance in the later molecular pathology study of heart failure, which may help to assist diagnose early heart failure in the future. More importantly, it was for the first time to apply a nanoprobe for visually monitoring the changes of micropeptide in living cells and in frozen tissue sections, and the design concept of the aptamer-based nanoprobe can be extended for fluorescence detecting other micropeptides.

Heart failure (HF) is a serious condition in which the heart is contractile deficit and unable to pump enough blood to meet the body's needs.1 In China, as in other countries, it is a common cause for hospital admission. Despite advances in cardiovascular treatments, HF still carries substantial risk of morbidity and mortality.2 At present it is easy to recognize HF in its moderate or severe stage when the patient has pronounced symptoms and signs accompanied by left ventricular (LV) systolic dysfunction, abnormal lung sounds, and higher brain natriuretic peptide (BNP) levels in the blood.2,3 However, due to compensatory mechanisms, early stages of HF always lack specific signs. But when compensation has been exhausted, the change in geometry of the ventricle leads to the impairment of function and the development of symptoms.4,5 Therefore, early diagnosis is widely believed as the key to treatment of HF, and the therapeutic effect is also strongly associated with the stage of HF at the time of diagnosis.6 During the last decade, there was accumulating evidence that disrupted calcium homeostasis is a major contributor to the pathophysiology of HF.7,8 While changes of other components contribute, it is generally agreed that much of the contractile deficit is due to reduced Ca2+ transients in myocyte, which controlled contraction and relaxation of heart muscle.9 In recent years, a growing body of evidence indicates that socalled long noncoding RNAs (lncRNAs) could produce a family of short peptides from small open reading frames. These short bioactive peptides called micropeptides had proven critical in the maintenance of calcium homeostasis in mammalian muscles by regulating activity of the sarcoplasmic

reticulum calcium ATPase (SERCA) calcium pump.10,11 These findings were thought to open new ways to understand cardiac function and pathologies.12 Phospholamban (PLN) is one of micropeptides that exists in cardiomyocytes sarcoplasmic reticulum (SR) as an active monomer of 52 amino acids. PLN sequence contains a cytoplasmic (residues 1-30) and a transmembrane segment (residues 31-52).13 PLN (unphosphorylated form) physically interacts with SERCA type 2a (SERCA2a) to antagonize its function, which returns cytosolic Ca2+ into SR after each contractile cycle in preparation for subsequent excitation-contraction events. Phosphorylation of PLN (p-PLN) at Ser16 or Thr17 reduces its affinity for SERCA2a, thereby increasing SERCA2a activity.14 Increased PLN expression has been clinically associated with human HF.15,16 Given the vital physiological effects of PLN in the process of HF, monitoring the dynamic changes of PLN in cardiomyocytes will be of great importance to evaluate the stage progression of HF and provide dependable information for early diagnosis. Currently, the mainly used methods to analyze the changes in PLN levels in cardiomyocytes are based on traditional techniques such as transfection with PLNfluorescent protein,17 western blotting,18 or immunohistochemistry,19 which are complicated and timeconsuming processes. Moreover, the cell transfection, lysate or immobilization process used in these methods makes it impossible to study the dynamic changes and natural situation of PLN. Thus, it is important to develop a non-invasive method for detecting PLN in living cardiomyocytes. Recently, fluorescent probes with the synthesis predominance on

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selectivity, sensitivity, biotoxicity, especially real time visualization are regarded as powerful tools for the noninvasive detection of biomolecules. However, due to the small size and low intracellular expression, visual monitoring of micropeptides in living cells by fluorescent probe is still a challenge. So far as we know, no visible fluorescence sensing approach for detecting PLN in living cells under physiological condition has been reported. Therefore, the development of a fluorescent probe for visually monitoring of the changes of PLN micropeptide in cardiomyocytes is extremely important. More importantly, as more micropeptides are discovered and more physiological functions of micropeptides are identified, studies about micropeptide label probes may hold the key to more biologically relevant discoveries. Aptamer is an artificial oligonucleotide receptor derived from in vitro selection with high specificity and affinity for a given target. The selected aptamers can recognize a wide variety of targets, including small molecules, proteins, cells and tissues by folding into well-defined three-dimensional shapes via the intramolecular interaction.20 Compared with antibodies, aptamers have low molecular weight, fast tissue penetration, high stability and low immunogenesis,21 and are thus considered great potential detection tools in analytical chemistry22, 23, 24. The PLN-specific RNA-aptamer we chose showed a high affinity for the cytoplasmic region of PLN, and was also able to distinguish PLN from its phosphorylated form (inactive state).25 Based on the above characteristics, this aptamer labeled with the fluorophore should be an ideal approach for analysis and visualization of PLN in living cells. However, an aptamer-based system capable of entering cells and remaining active in the intracellular environment has been proven to be intractable, as it is difficult to affect the cellular uptake of nucleic acid-based compounds without additional transfection reagent. Moreover, oligonucleotides can become unstable and degraded in cellular environments, and thus their application in vivo has been challenging.

Scheme 1. Nanoprobe’s transmemberane, working mechanism illustration, and detection of PLN in cardiomyocytes. Herein, we applied 13 nm Au nanoparticles (AuNP) to design and synthesize an aptamer-based near-infrared fluorescence nanoprobe, by functionalizing thiol-modified short DNA strands (C-strand). The C-stand is connected to the surface of AuNPs via the gold–thiol bond. The RNA-aptamers labeled a fluorophore (Cy5) can partly hybridize to the C-strand (the details of the sequences and dyes are shown in Table S1). In the bound state, the fluorescence of Cy5 was quenched by AuNPs, and the dense shell of oligonucleotides is expected to enhance the stability of the nanoprobe and improve the uptake of cells to the nanoprobe. When the nanoprobe enters the cardiomyocytes, the aptamer in the nanoprobe is capable of specifically binding the cytoplasmic segment of PLN, causing a spontaneous

interchain separation of the C-strand and the Cy5-aptamer. The fluorescence of Cy5 then recovers and its fluorescence intensity is correlated with the relative amount of PLN. The details of this approach are shown in Scheme 1. As expected, the nanoprobe can rapidly and selectively respond to PLN in vitro. Furthermore, using this nanoprobe, we successfully observed the significant differences in the expression of PLN in living cardiomyocytes treated with chloroquine and metformin, and in frozen tissue sections of heart from HF mice. EXPERIMENTAL SECTION Isolation of Primary Cultured Mouse Neonatal Cardiomyocytes. Primary cultured mouse neonatal cardiomyocytes (CMNCs) were isolated and cultured according to the previous protocol of Stephan Lange et al, which is summarized as follows.26 1-3 days old neonatal C57BL/6 mice were rinsed quickly in 75% ethanol solution for surface sterilization. Hearts were extracted from the body with curved scissors and transferred immediately into the bacterial dish containing phosphate buffer saline (PBS, 0.01 M, without Ca2+, Mg2+) on ice. The hearts were washed in the PBS (0.01 M, without Ca2+, Mg2+) for three times to remove blood and minced into small pieces (approximately 0.5-1 mm3, or smaller) using the curved scissors. The minced hearts were transferred into a conical tube containing 10 ml of the isolation medium (on ice), and incubated with gentle agitation at 4 °C overnight. The tissue fragments were sunk to the bottom of the tube and the supernatant was removed. 5 ml of digestion medium and 5 ml of collagenase/dispase mixture were added to the tissue fragments, and the mixture were incubated at 37 °C with gentle agitation for 20-30 min. Then the tissue fragments were gently triturated using a pre-wetted 10 ml cell-culture pipette for about 10-20 times. The tissue fragments should mostly disperse during this step, releasing the cells into suspension. Conical tube containing suspended cardiomyocytes was centrifuged for 5 min at 300 rpm. The supernatant and plate cells were remove into a 10 cm cell culture dish and incubated for 1-3 h in cell culture incubator in order to remove fibroblasts and endothelial cells, which will adhere to the uncoated cell-culture dish. After incubation, non-adherent cardiomyocytes were washed from the 10cm dish and cultured in confocal dishes. Adriamycin-induced HF model. 4 weeks old C57BL/6 mice were treated with an intraperitoneal injection of Adriamycin (2 mg/kg) 11 times over 7 weeks (total dosage: 22 mg/kg) in order to prepare an adriamycin-induced cardiomyopathy model. Prior to each drug administration, the body weight of the animal was measured for recalculating dosage. In addition, a control group of normal mice of the same age were injected with the same volume of saline. Then mice in both groups were normally fed until 10 weeks. After sacrifice, the hearts of mice were collected for the follow-up experiments. RESULTS AND DISSCUSSION Confirmation of the specific binding of Cy5-aptamer to PLN. As the specific binding of aptamer to target molecule is critical for the fluorescence recovery of the nanoprobe, we first verified the binding capacity of the PLN-specific RNA-aptamer labeled with Cy5 to PLN by aptamer binding assay. The result showed that the Cy5-aptamer group showed an obvious dosedependent fluorescence signals compared with the blank and scramble Cy5-RNA group (Figure S1), which indicated that the

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ACS Sensors aptamer we chose, even labeled with Cy5, can still specifically bind to PLN. Synthesis and characterization of the nanoprobe. Since the fluorescence signals of most dyes could be quenched by the nanoparticles within a certain distance,27,28 the C-strand hybridized with PLN-specific RNA-aptamer labeled with Cy5 was assembled on the surface of AuNPs (13 nm) by the Au–S bond. A visible color change can be observed between the AuNPs and nanoprobe solution (Figure 1A). And transmission electron micrographs exhibited that the morphology of the nanoprobe after nucleotide sequence modification did not change obviously compared to the naked AuNPs, and both of them displayed spherical morphology with an average size of 13 nm (Figure 1B). For the UV-Vis absorption spectra, the maximum absorption peak was red-shifted from 521 nm to 525 nm after the functionalization of the nanoprobe (Figure S2).The above results suggested the successful assembly of Cy5aptamer on the surface of AuNPs. Subsequently, the loading capacity of Cy5-aptamers were quantified according to the previous fluorescence quantitative method.29,30 The statistic results revealed that each AuNP carries an average of 246 ± 2 Cy5-aptamers (Figure S3).

A

manner, and the analysis demonstrated a good linear correlation (R = 0.9914) between the fluorescence intensity and the concentration of PLN, ranging from 0 to 200 M. The regression equation was F=35.722[PLN] M -66.52. Based on the above data, we calculated the detection limit of the nanoprobe which was as low as 181 nM.

Figure 2. Fluorescence responses of the nanoprobe toward PLN. Dynamic studies (A) and the spectroscopic property (B) of the nanoprobe (1 nM) responded to PLN (100 M). (C) Fluorescence spectra of the nanoprobe (1 nM) after incubation with various dosages of PLN (0-200 M) for 1 h (λex/λem = 642 nm/666 nm). (D) Linear fitting of PLN-dependent fluorescence intensity at 666 nm.

B

Figure 1. Preparation and characterization of nanoprobe. (A) Photograph and (B) transmission electron micrographs of AuNPs and nanoprobe. Scale bar = 50 nm. Responses of the nanoprobe toward PLN in vitro. After the nanoprobes’ physical characterizations, we next attempted to optimize the fluorescence measurement conditions, including reaction time, pH and ionic strengths. First, the dynamic response of the nanoprobe to PLN was examined. Figure 2A showed that the fluorescence intensities of the reaction solution were significantly enhanced with increasing processing time, and reached plateau within 30 min. The excitation/emission wavelengths were set as 642/666 nm (Figure 2B). The results validated that the nanoprobe owned an excellent fluorescence recovery in a short time, which benefited from the short binding time of aptamer to PLN. Then the influences of pH values and ionic strengths on the response of nanoprobe were investigated, respectively. As shown in Figure S4 and Figure S5, the data demonstrated that the nanoprobe was stable and can perform its best function under physiological condition (pH 7.4, PBS 0.01 M). Taken together, these results implied that the nanoprobe held a desired ability to response to PLN within 30 min under physiological condition, which makes it an optimal candidate for application in cells. On this basis, the responses of the nanoprobe to various concentrations of PLN were measured under simulated physiological condition. As shown in Figure 2C and 2D, the fluorescence intensity of the reaction solution increased by addition of the synthetic PLN in a concentration-dependent

Reaction Selectivity of the nanoprobe in vitro. To determine the reaction selectivity of the nanoprobe, various potential interfering species, such as GSH, glucose, amino acids, Na+, K+, Ca2+, OH, ClO-, H2O2, H2S, NO, vitamin C (Vc), fetal bovine serum (FBS) and other two micropeptides of the same family, sarcolipin (SLN)31 and myoregulin (MLN)10, were examined in parallel under the same conditions. As shown in Figure 3, the fluorescence intensity of the nanoprobe exhibited a significant increase in the presence of PLN, while other molecules mentioned above, even FBS, did not give rise to a notable fluorescence recovery. This result indicated that the nanoprobe should present high selectivity toward PLN in vivo.

Figure 3. Selective studies of the nanoprobe for PLN. The fluorescence was measured with 666 nm after mixing the nanoprobe (1 nM) with PLN (100 M), SLN (100 M), MLN (100 M), FBS (0.1% v/v) and other potential interfering species (1 mM) for 1h, λex/λem = 642 nm/666 nm.

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Nuclease stability and cytotoxicity of the nanoprobe. Before applying the nanoprobe for detecting and imaging PLN in living cells, the nuclease stability of the nanoprobe was evaluated under simulated physiological condition. As the nanoprobe possessed both DNA and RNA sequences, the enzyme deoxyribonuclease I (DNase I) and ribonuclease I (RNase I) were used to determine the nuclease stability of the nanoprobe, respectively. In Figure 4A, comparing with the blank control group, the nanoprobes treated with either DNase I (2 U/L) or RNase I (5 g/ml) were not detected with a significant fluorescence signal change in 2 h. A better stability and resistance against nucleolytic degradation should benefit from the phosphorothioate modification of every adenosine of the Cy5-aptamer.25 However, the three groups of solutions’ fluorescence intensities all displayed significant enhancement after 200 M of PLN addition. Hence, the fluorescence recovery via specific binding of the aptamer to PLN was further confirmed. For the next step, the investigation of the nanoprobe’s cytotoxicity was carried by the 3-(4,5-dimethylthiazol-2)-2,5diphenyltetrazolium bromide (MTT) assay in cardiomyocytes line H9c2. As shown in Figure 4B, both the nanoprobe (1 nM) and AuNPs had a negligible effect on H9c2 cells viability up to 48 h, demonstrating the nanoprobes’ low cytotoxicity on living cells and potential applications in biological samples.

Figure 4. Nuclease Stability and cytotoxicity of the nanoprobe. (A) The nanoprobes’ fluorescence intensities of (1 nM) addition of 5 g/ml RNase I (red trace) or 2 U/L DNase I (green trace) in 2h, compared with control group (blank trace). Insets: fluorescence recovery spectra after addition of PLN (200 M) to the above groups for 1h, respectively. λex/λem = 642 nm/666 nm. (B) Cell viability estimated by MTT assay. H9c2 cells were incubated with naked AuNPs (1 nM, orange trace), nanoprobe (1 nM, red trace) and PBS as control (0.01M, black trace) for 48 h, respectively. Imaging of PLN in vitro. The nanoprobe was then applied to simultaneously image PLN in primary cultured mouse neonatal cardiomyocytes (CMNCs), which had both been used to investigate PLN.32,33 Confocal laser scanning microscopy (CLSM) was applied to study the PLN via a red fluorescence channel. And as shown in Figure 5A and 5B, the fluorescence intensity distinctly enhanced in CMNCs with the increasing time, and then reached a steady value at 2h. Similar fluorescence signals were also observed in H9c2 cells (Figure S6). Additionally, subcellular co-localization experiment revealed that the staining area by the nanoprobe was highly overlapped with the staining area labeled by a PLN fluorescent antibody, implying the nanoprobe mainly targeted PLN in cardiomyocytes (Figure S7). Previous researches indicated that chloroquine could increase PLN levels by inhibit lysosomes and conversely metformin will increase degradation of PLN via

autophagy.33 Based on the above, we performed three parallel experiments to further confirm the quantitative analysis function of the nanoprobe in living cells. As expected, both total PLN and p-PLN increased after chloroquine treatments and decreased by metformin (Figure S8), so the unphosphorylated PLN should follow the same trend. Consistent with the western blotting results, in our nanoprobe experiments, stronger red fluorescence was observed in CMNCs in response to chloroquine treatments. Metformin pre-processing lead to weakened fluorescence intensity compared with the control group (Figure 5C and 5D). Thus, these results together demonstrated that the nanoprobe was capable of detecting dynamic changes of PLN expression in cardiomyocytes under natural situation.

Figure. 5. In vitro CLSM images of endogenous PLN. (A) Fluorescence imaging of CMNCs after incubation with the nanoprobe (1 nM) for 0, 0.5, 1, 2, and 4 h at 37 °C. Scale bar = 75 m. (B) Quantitative analysis of fluorescence intensity in (A). (C) CMNCs were pretreated with chloroquine (100 M) or metformin (2.5 mM) for 24h, and incubated with the nanoprobe (1 nM) for 2 h at 37 °C. Scale bar = 75 m. (D) Quantitative analysis of fluorescence intensity in (C). Compared with the control groups, *: P＜0.05, **: P＜0.01, ***: P＜0.001. Imaging of PLN in frozen sections of mice heart tissue. Finally, in order to illustrate the nanoprobes’ feasibility of in vivo imaging PLN, the mice models of HF were established according to Teraoka's method.34 Compared to wild-type control mice, obvious body weight (BW) losses were presented in HF mice, while the heart/body weight (HW/BW) ratio showed a remarkable elevation (Figure S9A). The hearts were also subjected to histopathological examinations, and eosin staining results showed that severe myocardial arranged disorder, myocardial hypertrophy, and interstitial fibrosis were observed in HF mice hearts as compared with the control mice (Figure S9B). Additionally, Western blotting revealed that no significant change was observed for total PLN expression, while PLN phosphorylation was significantly decreased in HF mice compared with the control group, indicating that higher

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ACS Sensors PLN expression in the hearts of HF mice (Figure S9C). The above results suggested that the HF mice models were successfully established, and overexpressed PLN might be the molecular pathogenesis of adriamycin-induced HF. For lacking of enhanced permeability and retention (EPR) effect in heart, the nanoprobe was hard to be applied directly in vivo. As myocardial biopsy is common used in clinical examination, we attempted to apply the nanoprobe in tissue sections. To maximally preserve the antigens in their original state, the left ventricular tissues were removed immediately after sacrifice of the mice and used for preparing frozen sections. After immersing in PBS for 3 min, the frozen sections were stained with 1nM nanoprobe for 30 min. Fluorescence images of these stained tissues showed that stronger red fluorescence was observed in HF mice group compared with the control group, which was in agreement with the previous western blotting data (Figure 6). These results further confirmed that PLN was higher expressed in the hearts of HF mice, and the nanoprobe could be well used for monitoring changes of PLN in frozen section of heart tissues. Therefore, we believed that the developed nanoprobe should be of great significance in the later clinical molecular pathology diagnosis of heart failure.

The Supporting Information is available free of charge on the ACS Publications website. The specific binding of Cy5-aptamer to PLN, Peptides/oligonucleotides sequences, Standard curves of the Cy5labeled RNA-aptamer, pH and ionic strengths effect on nanoprobe, western blots assessing endogenous PLN expression in CMNCs, fluorescence images of PLN in H9c2 and CMNCs, and characterization of adriamycin-induced HF mice model (PDF).

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected]. Fax: 86531-86180017.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (21575081, 21775091, 21535004 and 91753111), and the Key Research and Development Program of Shandong Province (2018YFJH0502).

REFERENCES

Figure 6. Confocal fluorescence imaging of PLN in frozen section of mice heart tissue. (A) The nanoprobe (1nM) were incubated with frozen section of the left ventricular tissue from HF and control mice for 30 min at 37 °C. Scale bar = 75 m. (B) Quantitative analysis of fluorescence intensity in (A). Compared with the control groups, n = 6, *: P＜0.05.

CONCLUSIONS In summary, we have designed and synthesized a novel fluorescent nanoprobe that can be used for detecting and imaging of PLN micropeptide in cardiomyocytes. The experimental results demonstrated that the nanoprobe had excellent performance in the detection of PLN with satisfying sensitivity, selectivity, nuclease stability and biocompatibility. Notably, the nanoprobe can be applied to visually monitor the changes of PLN in living cardiomyocytes and in frozen section tissues. Combined with myocardial biopsy, the nanoprobe should be of great significance in the later clinical molecular pathology diagnosis of HF, which is beneficial in evaluating the stage of HF progression and in making treatment decisions. More importantly, our design concept of the aptamer-based nanoprobe could be extended for fluorescence detecting other micropeptides in the future.

ASSOCIATED CONTENT Supporting Information

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